Metastatic colorectal cancer (CRC) is one of the most deadly cancers, characterized by poor prognosis and low survival rate. The multi-kinase inhibitor regorafenib can improve patient survival and has recently been approved for the treatment of metastatic CRC. Regorafenib inhibits the RAS/RAF/MEK/ERK pathway, which is aberrantly activated in most CRCs due to prevalent RAS and RAF mutations. However, the anticancer mechanism of regorafenib is not well understood. No genetic alteration has been linked to regorafenib sensitivity or resistance in CRC cells. The sometimes serious side effects of regorafenib observed in clinical studies make identifying responder patients a critical mission of precision medicine, to avoid unnecessary treatment. Our preliminary studies reveal that the therapeutic activity of regorafenib in CRC cells is associated with apoptosis induction and proteasomal degradation of Mcl-1, a pro-survival Bcl-2 family protein. CRC cells that are intrinsically insensitive to regorafenib lack Mcl-1 degradation and contain inactivating mutations of FBW7, a tumor suppressor and E3 ubiquitin ligase that is frequently mutated in CRCs. In response to regorafenib treatment, FBW7 binds to phosphorylated Mcl-1 and is required for its ubiquitination and subsequent degradation. Blocking Mcl-1 phosphorylation by a genetic knock-in approach abrogates its binding to FBW7 and degradation, and suppresses regorafenib-induced killing of CRC cells. Furthermore, CRC cells with acquired regorafenib resistance were found to have blocked Mcl-1 degradation and enrichment of FBW7 mutations. We hypothesize that FBW7-mediated Mcl-1 degradation is crucial for the anticancer effects of regorafenib and that FBW7 mutations disrupt regorafenib-induced Mcl-1 degradation, resulting in regorafenib resistance in CRCs. We propose to test this hypothesis and its translational implications by using genetic and biochemical approaches. Aim 1: Delineate how Mcl-1 degradation mediates the anticancer activity of regorafenib in CRC cells. Aim 2: Determine the effects of oncogenic mutations on regorafenib sensitivity and Mcl-1 stability. Aim 3: Elucidate the roles of FBW7 mutations and Mcl-1 stability in acquired resistance to regorafenib. In parallel with ongoing clinical studies on regorafenib, these studies may establish FBW7 mutational status and Mcl-1 stability as key determinants of regorafenib sensitivity in CRCs. They may also provide a rationale for effective combinations of regorafenib with other drugs, as well as new tools for investigating the broad functional roles of Mcl-1 and FBW7 in targeted anticancer therapies, and for overcoming therapeutic resistance caused by genomic instability in CRC cells.